Self-renewing, pluripotent human stem cells may provide a virtually unlimited donor source of neural progeny for far reaching applications. Neural differentiation of human pluripotent stem cells may provide access to study all steps of human neurogenesis, and therefore may serve as an in vitro model for the discovery of new genes and the development of new drugs. Neural progeny derived from human pluripotent stem cells may serve for testing and high throughput screening of molecules for neurotoxic, teratogenic, neurotrophic, neuroprotective and neuroregenerative effects. Human pluripotent stem cells may serve as an unlimited source of neural cells for transplantation and gene therapy of neurological disorders (1-3).
Reubinoff et. al. pioneered the development of highly enriched cultures of developmentally competent early neural precursors (NPs) from human embryonic stem cells (hESCs) (4). In addition, Itsykson et al demonstrated the controlled conversion of hESC into NPs in chemical defined culture conditions, in the presence of the BMP antagonist noggin (5).
The potential involvement of the Notch signaling pathway and the γ-secretase complex in pluripotent cell differentiation has been suggested by several groups. Notch signaling can be modulated by altering the activity of the γ-secretase complex. The proteins which form this complex include Presenilin, nicastrin, aph-1, pen-2 and related proteins(14). γ-secretase inhibitors were shown to reduce the level of Notch signaling.
Condie et al. (18) describes compositions and methods for the stabilization of pluripotent cells in an undifferentiated state and reduction of percentage of spontaneously differentiated cells in the pluripotent cell culture, by inhibition of components of the γ-secretase complex. On the other hand, incubation of the cells with an activator of Notch signaling resulted in differentiation of the cells into neuronal cells. Condie et al. (18), and also Lowell S. et. al. (12), describe a role for the Notch signaling system in controlling the fate of pluripotent stem cells. Accordingly, activation of Notch signaling in ES cells directs their differentiation towards the neural lineage.
Similarly, also LOWELL et al. (19) disclose that activation of Notch signaling in ES cells promotes differentiation towards a neural fate.
Wang S et al. (15), and Givogri M I et al. (16) discuss the potential role of the Notch system in oligodendroglial differentiation during development. John et al. (17) discuss the potential role of Notch system in controlling remyelination in MS. None of these studies however was performed in humans, or in ES cells, or in ESC-derived neural progenitors. Also, none of these studies concerns spinal cord development.
Differentiation of human embryonic stem cells towards motor neurons (also known as motoneurons) have been previously described (6-8). However, the efficiency of directing hESC to a motor neuron fate was limited and only 20-50% of the cells in differentiated cultures expressed markers of motor neurons. Differentiation towards a motor neuron fate was mainly induced by two factors. Retinoic acid was used for caudalization and sonic hedgehog (SHH) or its agonist purmorphamine for ventralization.